Life-threatening necrotizing group A streptococcal (GAS) infections occur in civilians and veterans of all ages worldwide. Mortality remains at 30-85% and major surgery, frequently including multiple limb amputations, is required in 50% of cases. Survivors require prolonged hospitalization and rehabilitation. Over half of all GAS necrotizing infections have no known portal of entry, yet infection becomes established at sites of non-penetrating minor injury such as a muscle strain. Patients with these cryptic infections are often prescribed non-steroidal anti-inflammatory drugs (NSAIDs) and clinical epidemiological studies have demonstrated a relationship between NSAID use and development of necrotizing GAS infections after injury. We have shown that injury of cultured human skeletal muscle cells increases the binding of invasive GAS and that surface-expressed skeletal muscle vimentin mediates this adhesion. Using a murine model of eccentric contraction (EC)-induced muscle injury, we have also shown that muscle strain upregulates vimentin gene expression and significantly increases the binding of GAS at the injured site - a process greatly augmented by administration of NSAIDs. Muscle regeneration after injury is mediated by vimentin-rich muscle cell precursors (satellite cells) and inflammatory cells (macrophages). Infiltrating macrophages undergo phenotypic and functional changes in response to stimuli produced by injured muscles and activated satellite cells. These altered macrophages then orchestrate muscle regeneration. NSAIDs suppress inflammation and directly delay muscle regeneration. Thus, we hypothesize that NSAIDs predispose to severe GAS infection by altering macrophage phenotype switching which in turn delays myogenesis, prolongs vimentin expression and impairs bacterial clearance. The Specific Aims of this proposal are: 1. To determine the mechanism by which NSAIDs enhance GAS infection of injured muscles. 2. To determine the direct and indirect effects of NSAIDs on the cellular interactome controlling vimentin expression in regenerating injured muscles. 3. To determine if NSAIDs directly alter macrophage phenotype switching and whether macrophages focused on muscle repair have reduced ability to ingest and/or kill GAS in the presence of NSAIDs. 4. To confirm trigger factor's role as the principal GAS vimentin binding adhesin. Impact on Veterans Health Care: GAS myonecrosis remains an important cause of morbidity and mortality among veterans and others. An understanding of the mechanisms by which NSAIDs augment in this host/pathogen interaction may lead to novel diagnostic, preventative and therapeutic strategies to reduce the severity of infection, shorten hospital stay and improve outcome in patients with invasive GAS myonecrosis. PUBLIC HEALTH RELEVANCE: Potential Impact on Veterans Health Care: Group A streptococcal (GAS) necrotizing fasciitis/myonecrosis is an important cause of morbidity and mortality in civilians and veterans worldwide. Survivors require prolonged hospitalization and rehabilitation, costing ~$90-180 million/yr. Over half of all GAS necrotizing infections occur at sites of minor injury (e.g., muscle strain) that do not break the skin. Patients with these cryptic infections are often prescribed non-steroidal anti-inflammatory drugs (NSAIDs) to alleviate the associated pain. However, several studies have demonstrated a relationship between NSAID use and more severe infection with worse outcomes. Muscle injuries, ranging from mild strains to those requiring amputation, are common among veterans, including active duty personnel, and inpatients in VA hospitals and nursing homes. Understanding the effects of NSAIDs in this host/pathogen interaction may lead to novel diagnostic, preventative and therapeutic strategies to reduce the severity of infection, shorten hospital stay and improve outcomes.